Over the last decade, femtosecond lasers have emerged as an important tool to perform accurate and fine dissections with minimal collateral damage in biological tissue. The most common surgical procedure in medicine utilizing femtosecond laser is LASIK. During the femtosecond laser dissection process, the corneal collagen fibers inevitably undergo biomechanical and thermal changes on a sub-micro- or even a nanoscale level, which can potentially lead to post-surgical complications. In this study, we utilized helium ion microscopy, complemented with transmission electron microscopy to examine the femtosecond laser-induced collagen fibrillar damage in ex vivo human corneas. We found that the biomechanical damage induced by laser etching, generation of tissue bridges, and expansion of cavitation bubble and its subsequent collapse, created distortion to the surrounding collagen lamellae. Femtosecond laser-induced thermal damage was characterized by collapsed collagen lamellae, loss of collagen banding, collagen coiling, and presence of spherical debris. Our findings have shown the ability of helium ion microscopy to provide high resolution images with unprecedented detail of nanoscale fibrillar morphological changes in order to assess a tissue damage, which could not be resolved by conventional scanning electron microscopy previously. This imaging technology has also given us a better understanding of the tissue-laser interactions in a nano-structural manner and their possible effects on post-operative wound recovery.